The first project area explores metabolic pathways that have been proposed based on in vitro studies to be important in non-replicating (NR)-MTb. We are exploring the importance of the biosynthesis of the cofactors biotin, coenzyme A and pyridoxal, peptidoglycan turnover, the role of putative F420-binding and genetically annotated pyridoxal-generating enzymes, beta-oxidation and iron acquisition and validating these by chemical and genetic means in non-replicating (NR)-MTb. We are continuing our studies to understand the role of the deazaflavin cofactor F420 in metabolism of this pathogen. We have identified several proteins that use F420 for unique redox reactions. In several of these, menaquinone can be reduced to its quinol by these enzymes in an F420-dependent manner which suggests that F420 may play a role in respiration of this pathogen reminiscent of F420-dependent respiratory complexes in methanobacteria. The substrate of some of these F420-binding proteins is also been identified by fragment-based approaches where we are identifying the binding affinity of commercially available metabolites to these proteins with and without bound F420. Small molecules that bind in the presence of reduced F420 are further characterized after incubation with the reduced cofactor and protein to determine whether these are enzymatically reduced. Finally, co-crystal structures of small ligands of interest bound to F420-protein complexes are determined with the ultimate goal of using the ligand-binding, enzymatic and crystallographic data to determine the true substrate of the proteins as well as enabling potential design of high-affinity inhibitors that can be used to probe the importance of the identified process in cellular metabolism. We are continuing our systematic analysis of potential bottlenecks in the coenzyme A metabolic pathway. The gene for each enzymatic step has been genetically manipulated in the pathogen to generate a strain where its expression is controlled by anhydrotetracycline levels in the culture media. In this way, we have identified those genes in the pathway that are permissive to fluctuation in their mRNA levels whereas repression of other coenzyme A biosynthetic genes have led to strains with poor growth or genetic revertants of the transcriptional regulatory systems suggesting that these may be chokepoints in coenzyme A biosynthesis. The metabolic changes in these cells with particular emphasis on metabolties in the coenzyme A super-pathway are analyzed in these mutants in combination with inhibitors of pantothenate synthase or pantothenate kinase. The second major focus area of this project starts from a different perspective and uses compounds that have activity against targets in cell wall biosynthesis to identify vulnerable steps in assembly of the macromolecular peptidoglycan-arabinogalactan-mycolate complex. We have identified inhibitors of several of these steps including the epimerase required for ribose-arabinose epimerization, synthesis of the peptidoglycan component, export of the mycolate precursors and inhibitors of the mycolyl transferases and are assessing the enzymatic activities associated with each step as well as their validation in terms of innate drug target potential during in vivo relevant growth and persistence of the organism. The third major focus of this project involves global approaches to understanding the metabolism in NR-TB. WE had previously demonstrated that reduction of fumarate to succinate plays a key role in reoxidation of reduced cofactors under hypoxic conditions. Although fumarate reductase/succinate dehydrogenase plays the key role in this process, MTb has 3 homologs of this enzyme complicating efforts to design inhibitors of this step in the organism. However, the interconversion of malate and fumarate precedes this step in the reductive pathway and is encoded by a single essential enzyme raising the potential of fumarase as a drug target under hypoxic survival of the pathogen. We have used the crystal structure of pyromellitic acid bound to the E. coli bound fumarase and used this to dock into the corresponding MTb fumarase crystal structure to generate a pharmacophore model of a MTb fumarase active site inhibitor. This pharmacophore model was used to screen 3 million compounds from the ZINC library to identify potential high affinity binders. The most promising of these were confirmed for their in silico ability to bind to the fumarase and subsequently these and their analogs were synthesized. The synthesized compounds were evaluated for their ability to bind to and enzymatically inhibit the MTb fumarase. The most promising of these were found to kill Mtb surviving under anaerobic conditions in a target-specific manner. Co-crystal structures of these inhibitors bound to fumarase were generated and are currently being refined to allow us to design MTb-specific fumarase inhibitors to validate the importance of this pathway in vivo. The reductive branch of the TCA cycle plays a role in reoxidizing reduced cofactors but the processes that play a role in maintaining the membrane potential of the organism under hypoxia remain poorly understood. We are using inhibitors of ATP synthesis and respiration to gain a better understanding of the interplay between respiratory, fermentative and energy generating processes under hypoxic growth and survival of the pathogen. In a fourth approach, we are identifying inhibitors of metabolism by high-throughput screening approaches performed under a variety of in vivo relevant environmental conditions. Hits from these screens have provided a useful tool to map metabolism of MTb as a function of carbon source, oxygen concentration or presence of low pH in the presence or absence of nitrosative stress and are currently being studied to identify the target. In the process of target identification, parallel studies are done to rapidly progress the hits to in vivo proof of concept studies so that the importance of the target for in vivo pathogenesis can be validated early on in the drug discovery process. One class of compounds were found to target folate biosynthesis. To further understand the vulnerable steps in folate biosynthesis we performed metabolomics of MTb exposed to a variety of inhibitors of folate metabolism and enzymatic analyses of the corresponding proteins in this pathway. We reported that para-aminosalicylic acid, a second line drug for the treatment of tuberculosis, poisons this pathway by acting as a substrate analog that gets incorporated into the folate pathway hereby poisoning folate pools. Other analogs of para-aminosalicylic acid are similarly incorporated but their metabolic fate depends on their chemical structure. The metabolic consequences of inhibitors of folate biosynthesis are being explored to understand the vulnerability of folate-dependent enzymes in this pathogen.